Prediction Model for Aneuploidy in Early Human Embryo Development Revealed by Single-Cell Analysis
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ARTICLE Received 26 Sep 2014 | Accepted 22 May 2015 | Published 7 Jul 2015 DOI: 10.1038/ncomms8601 OPEN Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis Maria Vera-Rodriguez1,2,3, Shawn L. Chavez1,2,w, Carmen Rubio3,4, Renee A. Reijo Pera1,2,w & Carlos Simon2,3,4 Aneuploidies are prevalent in the human embryo and impair proper development, leading to cell cycle arrest. Recent advances in imaging and molecular and genetic analyses are postulated as promising strategies to unveil the mechanisms involved in aneuploidy generation. Here we combine time-lapse, complete chromosomal assessment and single-cell RT–qPCR to simultaneously obtain information from all cells that compose a human embryo until the approximately eight-cell stage (n ¼ 85). Our data indicate that the chromosomal status of aneuploid embryos (n ¼ 26), including those that are mosaic (n ¼ 3), correlates with significant differences in the duration of the first mitotic phase when compared with euploid embryos (n ¼ 28). Moreover, gene expression profiling suggests that a subset of genes is differentially expressed in aneuploid embryos during the first 30 h of development. Thus, we propose that the chromosomal fate of an embryo is likely determined as early as the pronuclear stage and may be predicted by a 12-gene transcriptomic signature. 1 Institute for Stem Cell Biology and Regenerative Medicine, Center for Reproductive and Stem Cell Biology, Stanford, California 94305, USA. 2 Department of Obstetrics and Gynecology, Institute for Stem Cell Biology and Regenerative Medicine, Center for Reproductive and Stem Cell Biology, Stanford, California 94305, USA. 3 IGenomix, Parc Cientific Universitat de Valencia, Catedratico Agustin Escardino 9, Paterna 46980, Valencia, Spain. 4 Department of Obstetrics and Gynecology, Fundacio´n Instituto Valenciano de Infertilidad, University of Valencia, INCLIVA Health Research Institute, Paterna 46980, Valencia, Spain. w Present addresses: Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Beaverton, Oregon 97006, USA. Department of Obstetrics and Gynecology, Oregon Health and Science University, Beaverton, Oregon 97006, USA or Department Physiology and Pharmacology, Oregon Health and Science University, Beaverton, Oregon 97006, USA (S.L.C.). Department of Cell Biology and Neurosciences, Montana State University, Bozeman, Montana 59717, USA or Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA (R.A.R.P.). Correspondence and requests for materials should be addressed to C.S. (email: [email protected]). NATURE COMMUNICATIONS | 6:7601 | DOI: 10.1038/ncomms8601 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8601 ver the last 20 years, the number of assisted reproduction stages were measured for each embryo unless removed for procedures has drastically increased and is expected to molecular or chromosomal analysis before reaching this stage of Ocontinue to do so as parenthood is postponed. According development. We began by examining previously reported to European statistics, more than half a million in vitro parameters including the duration of the first cytokinesis, the fertilization (IVF) cycles are performed annually, resulting in time between the two- and three-cell, and time between the three- 100,000 newborns or 1.5% of all babies born in Europe1. and four-cell stages2,3 (Supplementary Table 1). The median Concomitant with the increased use of assisted reproduction, duration of the first cytokinesis was 20 min (range 15 min to there is an increase in the number of studies that have 2.9 h); the time from two- to three-cell stage was 11.4 h (range investigated the dynamics of human embryo development. 0–16.8 h); and the median time between the three- and four-cell Time-lapse imaging provides a non-invasive alternative to the stage was 1.3 h (range 0–22.1 h). Besides evaluating previously static morphological assessment of embryos, allowing the identified imaging parameters, we also measured the time evaluation of embryo viability via the measurement of between pronuclei disappearance (PNd) and the start of the predictive parameters on the basis of developmental kinetics2,3. first cytokinesis, a recently described parameter that has been In addition, time-lapse allows the observation of cellular events linked to human embryo viability4,5, but not to the chromosomal that would otherwise remain undetected by conventional status. As shown in Supplementary Table 1, the median for the methods such as bipolar one- to three-cell divisions4,5, time between PNd and the start of the first cytokinesis was 2.7 h fragment reabsorption6,7 and blastomere fusion5. Technologies (range 15 min to 22.2 h), suggesting that the wide range of such as array comparative genomic hybridization (aCGH) have this parameter might reflect underlying differences in embryo facilitated the transition from the study of nine chromosomes developmental potential. using fluorescent in situ hybridization (FISH) to the analysis of all In addition to normal cell cycle divisions, we also were able to 23 chromosome pairs simultaneously in a single cell8. More detect abnormal division events in certain embryos, including recently, several studies have combined additional technologies in divisions from one to three cells or from one to four cells instead order to generate a ploidy prediction model on the basis of of the typical one to two cells. These atypical events were embryo kinetics7,9,10. observed in 23.6% (n ¼ 20) of embryos with the majority dividing Recent advances in the analysis of gene expression at the from one to three blastomeres (n ¼ 17; Supplementary Movie 1) single-cell level provide the opportunity to examine underlying and a much smaller subset dividing from one to four cells (n ¼ 3; molecular programmes involved in embryo ploidy generation. Supplementary Movie 2). Notably, 85% (n ¼ 17) of all abnormal Owing to technical limitations, previous studies of quantitative divisions occurred during the first mitosis, whereas only 15% reverse transcription PCR (RT–qPCR) of human embryo (n ¼ 3) occurred in either the second or third mitosis development analysed only a select group of genes11,12 and/or (Supplementary Movie 3), stressing the importance of the first large pools of embryos13, which can be confounded by potential mitotic division. Since irregular divisions produce a greater embryo heterogeneity. As RT–qPCR techniques have evolved and number of fast-dividing cells, the number of blastomeres was become more sensitive, gene expression analysis of individual disregarded as a method to stage the embryos. As an example, an human embryos followed3,14 and, more recently, single-cell embryo with four blastomeres typically results from three RT–qPCR analysis has become a reality3,15,16. To date, only a consecutive normal divisions; however, it could also be produced couple of studies have correlated gene expression patterns with during one direct cleavage from one to four cells. In both cases, aneuploidy in human embryos, one of which observed differential the embryos would be at the four-cell stage and it would be expression of certain epigenetic mediators in euploid versus inappropriate to similarly classify them. Thus, the time from PNd aneuploid embryos and the other examining DNA repair genes in was chosen as the starting reference point to define the embryo embryos with single over complex aneuploidies17,18. However, stage, since it was the first event observed post thaw and before the latter study did not include euploid embryos, only evaluated mitotic divisions. six chromosomes via FISH and analysed 15–20 pooled day-4 With regards to morphology, we also evaluated the incidence embryos rather than individual embryos or single cells. and timing of cellular fragmentation using multiplane imaging in Here we seek to examine the relationship between aneuploidy, order to obtain a three-dimensional model of each embryo and an embryo kinetics and transcriptome profiles at the earliest stages of accurate measurement of fragmentation degree. The vast majority the human preimplantation development. By simultaneously of embryos exhibited less than 25% fragmentation (n ¼ 62), assessing imaging behaviour, complete chromosomal composi- and no embryo showed fragmentation greater than 60% tion and the expression of B90 genes in single cells from whole (Supplementary Fig. 1a). Of the 64 embryos displaying fragmen- human embryos cultured at the one- to approximately eight-cell tation to any degree, most of them (68.8%) initially fragmented stage, we test the hypothesis that generation of aneuploidy may during the first division, 21.9% of embryos fragmented before and influence or be influenced by changes in gene expression and 9.4% after the completion of this division, further highlighting the embryo kinetics. Our results show that aneuploid embryos exhibit importance of the first mitosis (Supplementary Fig. 1b). altered developmental timing and a different transcriptomic profile than euploid embryos within the first 30 h of development. Thus, future studies should focus on the zygote as a key stage of Correlation between the ploidy status and developmental kinetics. human embryonic development and a potential source of non- Complete ploidy results were obtained for 89 cells from a total invasive biomarkers that can prospectively predict ploidy. of 57 embryos.